Emc102a

Sera were collected from the peripheral blood by centrifugation at 1,000 g. The concentrations of TNF-α, TGF-β, IL-2, IL-10, IFN-β, and IL-6 in peripheral blood were measured using ELISA kits according to the manufacturers' instructions [EMC102a, EMC002, EMC005, EMC107b, EMC004, EMC016 (NeoBioscience)]. Optical densities were measured at 450 nm using a microplate reader [ELX800 (BioTek)] and a standard curve was drawn to calculate the content of immune factors.

The RAW264.7 cells or SK-HEP-1 cells were seeded into 6-well plates at a density of 1 × 10⁶/well and cultured overnight, and the cells were stimulated with different concentrations of CnB for 24–48 h. The culture supernatant was collected and measured by ELISA (Cloud Clone, SEA222Mu; SEA563Mu; SEA133hu; SEA222hu or Neobioscience Technology Co., Ltd.; EMC102a, EHC143; EMC106) according to the manufacturers’ protocols.

Enzyme‐linked immunosorbent assay kits were used to measure the concentration of IL‐1β, IL‐6 and TNF‐α in supernatant of BMDM cells following the instruction of kits (IL‐1β [EMC001B], IL‐6 [EMC004] and TNF‐α [EMC102A], Neobioscience). The concentration of each cytokine was defined by the absorbance measured with a microplate reader at 450 nm.

Level of IL-1β (EMC001b, Neobioscience), IL-6 (EMC004, Neobioscience), IL-10 (EMC005, Neobioscience) and TNF-α (EMC102a, Neobioscience) in cell supernatants were measured. And CHIL3 levels in serum were measured using an ELISA kit (ELM-YM1, RayBio, GA, USA). According to the manufacturer's instructions. Added samples and standard into strips and incubate for 2.5 hours at room temperature with gentle shaking. Discard the solution and washed. Added 100 µl of prepared biotinylated antibody for 1 hour. Then reacted with prepared Streptavidin solution. TMB One-Step Substrate Reagent used. 45 minutes after chromogenic reaction, stop reaction was added and each well read at 450 nm immediately.

The levels of the inflammatory cytokine TNF-α, IL-1β and IL-10 in cell supernatants were measured using ELISA kits (cat. nos. EMC102a, EMC001b and EMC005, respectively; Neobioscience Technology Co., Ltd.) according to the manufacturer’s instructions.

The cerebral cortex and hippocampus tissues of PAP mice were homogenized in radio immunoprecipitation assay lysis buffer (P0013B, Beyotime, China) containing protease inhibitors (ST506, Beyotime, China), sonicated briefly, lysed on ice for 30 min with gentle agitation, and then centrifuged at 14,800 × g for 20 min at 4°C to remove debris. Aliquots of the lysates were tested for Aβ_1-42 monomers and Aβ oligomers, using the Human/Rat Beta Amyloid (42) ELISA kit (290–62601, Wako, Japan) and Mouse Aβ Oligomer ELISA kit (AB6094, Abmart, China), respectively, according to the manufacturer’s protocols. Plasma and brain tissue IL–1β and TNF–α were determined using the Mouse IL-1β (EMC001b, NeoBioscience, China) and TNF-α (EMC102a, NeoBioscience, China) ELISA kits in accordance with the manufacturer’s protocols. In addition, feces of PAP mice were homogenized at 50 mg/mL in sterilized PBS, and large debris were removed by brief pulsed centrifugation. The fecal suspension was centrifuged at 14,800 × g for 15 min at 4°C to remove sediments, and the levels of fecal LCN2 and albumin were assayed using the Mouse LCN-2/neutrophil gelatinase-associated lipocalin (NGAL) Quantikine ELISA kit (MLCN20, R&D Systems, USA) and Mouse Albumin ELISA kit (E13878m, Cusabio, China).

Cytokine levels in serum or conditioned media were detected using an ELISA kit following the manufacturer’s instructions, including LXA4, tumor necrosis factor alpha (TNF-α) (EMC102a; NeoBioScience, Shenzhen, China) and IL-1β (EMC001b; NeoBioScience, Shenzhen, China).